Qiao et al. AMB Expr (2018) 8:176 https://doi.org/10.1186/s13568-018-0706-0

ORIGINAL ARTICLE Open Access Egg immunoglobulin interactions with Porphyromonas gingivalis to impact periodontal infammation and halitosis Wu Qiao1, Fang Wang2† , Xiaochen Xu3, Shujun Wang4, Joe Mac Regenstein5, Bin Bao1,6* and Ming Ma1,6*

Abstract Porphyromonas gingivalis is a pathogenic Gram-negative anaerobic bacterium that colonizes the subgingival region of gums. These bacteria can invade periodontal tissues, form plaques, and produce volatile sulfur compounds (VSC) and volatile organic compounds (VOC). Egg yolk immunoglobulin (IgY) that was specifcally produced in egg after chickens were challenged with P. gingivalis could control and prevent oral diseases caused by P. gingivalis. The releases of P. gingivalis ofensive metabolic odors in vitro and in vivo were determined using a Halimeter and GCMS. With IgY bacterial growth was inhibited, and the relative amounts of VOC and VSC were decreased. The scores for the oral health index and the levels of IL-6 and TNF-α are also decreased. All treatment groups showed signifcant anti- infammatory efects, which strongly suggests that specifcally IgY against P. gingivalis may be an efective treatment for the prevention and protection of periodontal infammation and halitosis. Keywords: Egg yolk immunoglobulin, Porphyromonas gingivalis, Periodontal infammation, Halitosis, Volatile sulfur compounds, Volatile organic compounds

Introduction and Jenkinson 2000). Te results in About 700 diferent microbial species cause a number tissue destruction and loss of alveolar bone and connec- of diferent infections and infammation in the oral cav- tive tissue, eventually leading to periodontal infamma- ity (Kuramitsu et al. 2007; Tsuzukibashi et al. 2014). Te tion (Larsson 2017). VSC and VOC cause the unpleasant most common microbial oral diseases are periodon- odors, which are referred to as halitosis (Lee et al. 2014). tal infammation (Ke et al. 2016) and halitosis (Tanda Some approaches have been reported and applied et al. 2015). Tese diseases may be caused by various to avoid oral diseases caused by P. gingivalis. Tey anaerobic bacteria such as Streptococcus mutans, Por- included inhibition of cell growth and initial cell adhe- phyromonas gingivalis, Prevotella intermedia, and Fuso- sion, and degradation of odors emitted using antibacte- bacterium nucleatum (Zhen et al. 2008; Patra et al. 2014). rial agents, and polyclonal and monoclonal . P. gingivalis is one of the major that colonizes Honey as a therapeutic option in periodontitis treat- the subgingival region of gums. Tese bacterial can ment acted as an antibacterial against P. gingivalis, but produce compounds that contribute to dental plaque, did not inhibited the formation of bioflms in an in vitro acquired immune response and release of unpleasant study (Eick et al. 2014). P. gingivalis was found at a fre- odor, such as adhesins, hemin binding , quency of 35% in patients with chronic periodontitis proteinases, volatile sulfur compounds (VSC) and vola- and clinical isolates were highly sensitive to metroni- tile organic compounds (VOC) (Holt et al. 1999; Lamont dazole and tetracycline (Gamboa et al. 2014). However, antibiotic resistance attributed to the use of metronida- *Correspondence: [email protected]; [email protected] zole and tetracycline has become an increasing problem †Fang Wang—Co-frst author (Patil et al. 2013). Parenteral or intraoral administration 1 College of Food Science and Technology, Shanghai Ocean University, No. 999 Hucheng Ring Road, Pudong New Area, Shanghai 201306, China of KAS2-A1-specifc protected Full list of author information is available at the end of the article against the development of P. gingivalis-induced bone

© The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Qiao et al. AMB Expr (2018) 8:176 Page 2 of 12

resorption and inhibited proteolytic activity, binding to Materials and methods host cells/proteins and co-aggregation with other peri- Materials odontal bacteria (O’Brien-Simpson et al. 2016). VSC Tis study used IgY against P. gingivalis (25,600 titer, were produced primarily by anaerobic bacteria that 370 mmol/l, according to the manufacturer) from Maxam produced large amounts of proteinases, many trypsin- Ltd. (Shanghai, China), and P. gingivalis (ATCC33277) like (van den Velde et al. 2007). And oral malodors were was purchased from American Type Culture Collection reduced by inhibition of P. gingivalis growth and neu- (Manassas, VA, USA). All other chemicals were analytical tralization of VSC (Lee and Baek 2014). Streptococcus reagent grade or better from Chinese suppliers. thermophilus, a probiotic bacterium, inhibited growth Te simple preparing process of P. gingivalis as an anti- of P. gingivalis, and reduced P. gingivalis-producing gen: P. gingivalis was maintained on Brain Heart Infusion VSC emissions. Nevertheless, S. thermophilus showed Broth (BHI) supplemented with 10% sheep . Stable- diferent antibacterial activity with diferent culture- phase P. gingivalis was dissolved in PBS (0.05 M, pH7.4) conditions. A possible reason may be that P. gingivalis and stored at 4 °C. Te solution mixed with Freund’s can inhibit the activity of antibacterial agents of S. ther- adjuvant in a ratio of 1:1 was used as the . mophilus or has weak resistance for the antibacterial agent (Lee and Baek 2014). Growth inhibition Recently, chicken egg yolk immunoglobulin, referred Te artifcial saliva was prepared using a method to as immunoglobulin Y (IgY), has been shown to be a reported previously (Saunders et al. 2000; Bjorklund et al. specifc that was produced in egg yolks after 2011). Briefy, the artifcial saliva was supplemented with chickens were challenged with bacteria, , or mucin from porcine (Sigma Chemical Co., St. parasites. Tus, IgY could specifcally control and pre- Louis, MO, USA) 2500 mg/l. Te saliva also contained vent diseases caused by their corresponding antigen (Li KCl 1160 mg/l, NaHCO­ 3 375 mg/l, KH­ 2PO4 355 mg/l, et al. 2012). Tese may be a convenient and economical ­NH4Cl 235 mg/l, KSCN 220 mg/l, ­CaCl2 210 mg/l, urea alternative to antibiotics to give passive immunization 175 mg/l, ­MgCl2 45 mg/l, thiamine 0.007 mg/l, ribofavin (Horie et al. 2004; Chalamaiah et al. 2017). IgY against 0.05 mg/l, folic acid 0.0001 mg/l, nicotinic acid 0.03 mg/l, Pseudomonas aeruginosa therapy facilitated rapid bac- pyridoxine 0.6 mg/l, pantothenic acid 0.08 mg/l, biotin terial clearance and moderated infammation in P. 0.0008 mg/l, B12 (cyanocobalamin) 0.003 mg/l, vitamin aeruginosa lung infections. It may serve as an adjunct K (menaphthone) 0.015 mg/l, bovine serum albumin to antibiotics in reducing early colonization (Tom- (Sinopharm Chemical Reagent Co., Shanghai, China) sen et al. 2016). IgY against Solobacterium moorei sig- 25 mg/l, choline 15 mg/l, uric acid 10 mg/l, alanine nifcantly inhibited growth and bioflm formation of S. 3.3 mg/l, arginine 1.9 mg/l, aspartic acid 1.6 mg/l, glu- moorei and decreased the bacterial level in the oral cav- tamic acid 3.9 mg/l, glycine 8.9 mg/l, histidine 1.0 mg/l, ity of mice after infection with S. moorei (Lee and Baek leucin 2.9 mg/l, iso-leucine 2.9 mg/l, lysine 2.7 mg/l, 2014). IgY against P. gingivalis inhibited dental plaque methionine 0.03 mg/l, phenylalanine 2.9 mg/l, pro- formation (Hamajima et al. 2007) and hemagglutinat- line 0.2 mg/l, serine 2.1 mg/l, threonine 2.9 mg/l, tyros- ing activity of P. gingivalis (Tezuka et al. 2006) in vitro, ine 2.1 mg/l, valine 1.8 mg/l, creatinine 0.1 mg/l, and 5 which may be useful in developing passive immuniza- α-amylase 3 × 10 U/l (Sigma). It was sterilized by passing tion against periodontal diseases caused by P. gingivalis through a 0.22 μm-flter (50 ml, Termo Fisher Scientifc, infection. Shanghai, China). P. gingivalis were cultured in brain Because of rats’ periodontal anatomy similar to heart infusion broth (BHI) (Shandong Haibo Technol- humans they are often used in the study of periodontal ogy Information System Co., Ltd., Qingdao, Shandong, diseases (Helieh and David 2011). In addition, rodent China). Colonies were counted using a hemocytom- models refect the state of the periodontal more sensi- eter (Shanghai Refned Biochemical Reagent Instrument tively. Terefore, periodontal diseases commonly have Co., Ltd., Shanghai, China). Bacterial liquid (0.5 ml) was been induced by placing a bacterial plaque retentive added to 4.5 ml artifcial saliva at a fnal concentration of ligature in the gingival sulcus around the molar teeth in ­108 CFU/ml. Ten 10 μl of IgY at concentrations of 370, animal models (Breivik et al. 2000). Te purpose of this 7.4 and 0.0037 mmol/l (diluted using 0.9% NaCl) was put study was to evaluate the efect of IgY against P. gingivalis into 5 ml tubes and were identifed as the high-dose, mid- on the growth of P. gingivalis, emission of VSC and VOC dose, and low-dose group, respectively. Te mixtures in vitro and in vivo, the scores for oral health indexes and without IgY were used as a control. Tese tubes were the levels of IL-6 and TNF-α to determine if IgY might be placed into an anaerobic package (Anaero Pack, Mit- an efective treatment for the prevention of periodontal subishi Gas Chemical Co., Tokyo, Japan), and incubated infammation and halitosis caused by P. gingivalis. at 37 °C. ­OD600 of the suspensions was measured using Qiao et al. AMB Expr (2018) 8:176 Page 3 of 12

a spectrophotometer (T6, Persee Ltd., Beijing, China) at intervals (Hou et al. 2014). Te infected rats were ran- 2 h intervals (Hou et al. 2014). Te growth inhibition on domly divided into three groups (n = 3). After the last P. gingivalis with IgY treatment was measured as OD­ 650. infection, 6 rats were smeared using a syringe with difer- ent doses of 0.2 ml IgY against P. gingivalis solutions, and Measurement of VSC in artifcial saliva using a Halimeter three rats served as a control group to which 0.2 ml non- A portable sulphide monitor (Halimeter, Interscan Co., specifc IgY was applied each day in the oral cavity for up Chatsworth, CA, USA) was used to assess the VSC lev- to 28 days. IgY at concentrations of 370 and 7.4 mmol/l els. Samples in sequence connect to the sampling tube of (diluted using 0.9% NaCl) were identifed as the high- the Halimeter for 30 s according to the manufacturer’s dose and low-dose group, respectively. instructions and three diferent readings were recorded per sample from the instrument without external Measurement of VOC in the oral cavity using GCMS calibration. Te samples were obtained using swabs that were wiped for 30 s in the oral cavity. Ten the swabs were placed Measurement of VOC in artifcial saliva using GCMS at the bottom of a headspace vial with a screw top lid Te artifcial saliva was concentrated by putting 6 ml on (20 ml, Supelco, Bellefonte, PA, USA) and extracted with a Poly-Sery Hydrophile Lipophilic Balance Solid Phase a coated micro extraction fber (65 μm, PDMS/DVB, Extraction Cartridge (500 mg, Anpel, Shanghai, China) Supelco) for 30 min at 65 °C. Te desorption time of the followed by passing dry nitrogen (99.9%, Linde, Shanghai, extraction was 5 min according to the manufacturer’s China) through the cartridge for 2 min. Te sample was instructions. eluted with 1 ml methanol. Te gas chromatograph was a model: HP-6890N, inter- Oral test faced with a mass selective detector HP-5973 (Agilent, Santa Clara, CA, USA). A fused silica capillary column Te plaque index (PI), gingival index (GI), and halitosis index (HI) were measured before and after treatment. HP-5MS (60 m × 0.32 mm × 1 μm, Agilent) was used with helium (99.999%, Linde) as the carrier gas (1 ml/ Te PI and GI were recorded using a periodontal probe min). Te injection volume for each sample was 1 μl (Pancer et al. 2016; Liu et al. 2012). Briefy, the periodon- (Wang et al. 2018). Te temperatures used were 250 °C tal pockets or gingival crevices of the selected tooth were for the detector, and 50–250 °C for the column with a probed and graded as follows: Score 0: Gingival margin heating rate of 8 °C/min. Peak identifcations were done and gingival papilla (GM&P) are healthy, and no bleed- using the National Institute of Standards and Technol- ing is observed after a slight probe. Score 1: GM&P are ogy (NIST) electronic library (access provided by Agi- mildly infamed; and changes in color, absence of edema, lent). Te estimation of the approximate amounts of odor and occurrence of punctate hemorrhages are observed chemicals was based on the area under the peaks accord- after a slight probe. Score 2: GM&P are moderately ing to the software provided with the instruments and infamed; changes in color, severe edema, and bleed- the determination of each peak’s proportional area of the ing are observed after a slight probe while the blood is total observed peak areas. still in the gingival crevice. Score 3: GM&P are severely infamed; changes in color, severe edema, ulcer, and Animal studies bleeding are observed after a slight probe; or spontane- ous bleeding, and blood is observed fowing out of the Healthy male Wistar rats (n 9, weight 160 10 g) pur- = ± gingival crevice. Te HI was assessed by direct snif- chased from Slac Laboratory Animal Co., Ltd. (Shanghai, ing (score 0–4) done by only one person, and the scores China) were housed 3/cage upon arrival in 3 standard refected the degree of the odor (Hu et al. 2007). ventilated cages for 1 week before experimental proce- dures began. All rats had ad libitum access to the rat feed and water throughout all experiments and were main- IL‑6 and TNF‑α assays in serum tained on a 12 h light cycle with lights on at 8:00. Te Te blood samples were taken from the heart after cer- testing was performed between 8:00 and 16:00. Te use vical dislocation. Te serum was cleared from cellular of animals, ethical clearance, and the study protocol components of the blood by centrifugation at 12,000 rpm were duly approved by the U.K. Animals (Scientifc Pro- for 10 min at 4 °C and stored at − 80 °C until use. IL-6 cedures) Act and User Committee of Shanghai ocean and TNF-α levels were measured using an enzyme-linked University (China). Maxillary second molars of the rats immunosorbent assay following the manufacturer’s were ligated with 5-0 silk thread and inoculated with instructions (Nanjing Jiancheng Bioengineering Institute, freshly prepared P. gingivalis ­(106 CFU/ml). Te rats Nanjing, China). A blank hole without sample only add- were challenged with P. gingivalis eight times at 2-day ing chromogenic agent and terminated liquid were used Qiao et al. AMB Expr (2018) 8:176 Page 4 of 12

for zero. Optical density (OD) at 450 nm was read using a Microplate Reader (Corona Electric, Hitachinaka, Japan).

Statistical analysis Statistical analysis was done using SPSS 11.0 for Win- dows (SPSS, Chicago, IL, USA). Data were shown as mean ± standard deviation. Te student t-test was used to quantify data for the microbial experiments. While Mann–Whitney U test (a non-parametric statistical) analysis was used to quantify data for the animals experi- ments. Te diferences were considered signifcant at the level of p < 0.05.

Fig. 2 The VSC values of artifcial saliva. The VSC values were Results recorded directly as the readings from the Halimeter, and the unit is Efect of IgY on growth inhibition ppb (ug/l). **Means diferent at p < 0.01. p 0.007 vs. the control and = low-dose, p 0.006 vs. the control and middle-dose, p 0.003 vs. the Upon addition of IgY in artifcial saliva, bacterial cells = = formed complexes with IgY that precipitated to the bot- control and high-dose tom of the culture tubes, and the artifcial saliva became clear (Zhen et al. 2008). Bacterial counts increased rap- idly in the control (Fig. 1). However, the growth rate was Efect of IgY on VOC inhibition in artifcial saliva signifcantly (p < 0.05) inhibited by the addition of IgY VOC are products of P. gingivalis metabolism. Te with each response signifcantly stronger than from the major classes produced are: alcohols (A1), aldehydes previous level. (A2), ketones (A3), carboxylic acids (A4), esters (A5), phenyl compounds (B), nitrogen compounds (C), sul- fur-containing compounds (D), and alkanes (E) (Xia Efect of IgY on VSC inhibition et al. 2008). In the artifcial saliva, 16 chemicals were Te VSC values are shown in Fig. 2. All three doses detected (Table 1). Te relative contents of VOC were reduce VSC signifcantly (p < 0.05) from the control but reduced before and after treatment with IgY (Fig. 3). not signifcantly diferent from each other (p ≥ 0.05). Te reduction of hydroxylamine was highest compared with control. Te reductions of 3-bromo-5-chloro-1- tosyl-1H-pyrrolo[2,3-b] pyridine, benzeneacetalde- hyde, 1,4-dioxoperhydropyrrolo[1,2-a]pyrazine, methyl elaidate, 2,4-Di-tert-butylphenol, methyl hexadecanoate, and methyl stearate were also signifcant (p < 0.05). Te diference values between the control and treatment groups were multiplied by the corresponding proportion (Table 1, Fig. 4). Te proportion of VOC were diferent depending on themselves odor (Outhouse et al. 2006). Te relative content of 2,4-di-tert-butylphenol, hydrox- ylamine, 1,4-dioxoperhydropyrrolo [1,2-a] pyrazine, trans-13-docosenamide, 3-bromo-5-chloro-1-tosyl-1H- pyrrolo[2,3-b]pyridine, and undecane were also signif- cantly reduced suggesting that IgY could reduce VOC by inhibiting bacterial growth. Fig. 1 The inhibitory efect of IgY on the growth of P. gingivalis. P. gingivalis was cultured in artifcial saliva medium containing 0 (control), 0.0037 (low-dose), 7.4 (mid-dose) or 370 (high-dose) mmol/l Efect of IgY on VOC inhibition in the oral cavity of rats of IgY. Data were obtained using three independent experiments with three measurements in each experiment, and are shown as Data from GCMS suggested that the bad breath of rats mean SD (n 3). *Means diferent at p < 0.05, and **Means diferent mainly was due to 9 classes of compounds with 20 spe- ± = at p < 0.01. p 0.038 vs. the control and low-dose, p 0.016 vs. the cifc chemicals involved (Table 2). After treatment = = control and middle-dose, p 0.008 vs. the control and high-dose = with IgY for 4 wks, the relative contents of VOC were Qiao et al. AMB Expr (2018) 8:176 Page 5 of 12

Table 1 Chemical composition of VOC produced by P. gingivalis in artifcial saliva detected using GCMS No. Retention time CAS no Name Structure Class Proportion of odor (%)a

1 1.00–4.60 7803-49-8 Hydroxylamine HO NH2 Nitrogen compounds (C-1) 15 2 10.8 122-78-1 Benzene acetaldehyde Aldehydes (A2) 2

O 3 11.9 1120-21-4 Undecane Alkanes (E) 10

4 19.3 96-76-4 2,4-Di-tert-butylphenol OH Phenyl compounds (B-1) 15

5 22.9 19179-12-5 1,4-Dioxoperhydropyrrolo[1,2-a- O Nitrogen compounds (C-2) 15 pyrazine HN N

O 6 24.3 84-69-5 Diisobutyl phthalate Phenyl compounds (B-2) 1 O O

O 7 24.9 5129-60-2 Methyl 14-methylpentadecanoate Esters (A5-1) 2

O

O 8 24.97 112-39-0 Methyl hexadecanoate O Esters (A5-2) 2 O

Br 9 25 866-54-6 3-bromo-5-chloro-1-tosyl-1H- Cl Sulfur-containing compounds (D) 15

pyrrolo[2,3-b]pyridine N N O S O

10 25 2416-20-8 Hexadecenoic acid O Carboxylic acids (A4-1) 1 OH

11 25.4 57-10-3 Hexadecanoic acid O Carboxylic acids (A4-2) 1

OH

12 27 1937-62-8 Methyl elaidate O Esters (A5-3) 2 O

13 27.34 112-61-8 Methyl stearate O Esters (A5-4) 2 O

14 27.5 112-79-8 Elaidic acid Carboxylic acids (A4-3) 1 OH O 15 27.7 57-11-4 Stearic acid Carboxylic acids (A4-4) 1 OH

O 16 29 10436-09-6 trans-13-docosenamide O Nitrogen compounds (C-3) 15

H2N a According to the contribution to the VOC each chemical’s proportion represents its relative area under the peak without further calibration signifcantly reduced compared with control. Te rela- isohexenyl ketone, and isooctyl alcohol (Fig. 5). Te tively higher reductions occurred with hexanal, 3,4,4-tri- diference values between the control and treatment methylcyclopent-2-en-1-one, trans-2-octen-1-al, methyl groups were multiplied by the corresponding proportion Qiao et al. AMB Expr (2018) 8:176 Page 6 of 12

Fig. 3 The reduction of VOC in artifcial saliva. In artifcial saliva 16 chemicals were detected (Table 1) using GCMS. Negative numbers represent an increase. The contents of VOC reduction are shown as mean with SD error bars (n 3). *Means that the contents of the control group continued to = increase, while the contents of the treatment group decreased signifcantly after administration. The p values are, in order, 0.027, 0.042, 0.033, 0.036, 0.034, 0.021, 0.030, 0.027 compared the control with the treatment groups

the metabolites produced by P. gingivalis included VSC, aldehydes, ketones, carboxylic acids, esters, phenyl compounds, nitrogen compounds, and alkanes. After treatment with IgY, these metabolites were signifcantly reduced compared with control. Previous reports indi- cated that the main volatile molecules contributing to oral malodor included VSC, diamines, phenyl com- pounds, alcohols, alkanes, short-chain fatty acids, nitro- gen compounds, and ketones (Bollen and Beikler 2012; Krespi et al. 2006). In addition, hexanal was detected in the experiment associated with halitosis. However, it is likely according to two reports that the hexanal may be a reaction intermediate. Because hexanal can generate hex- anedione (Nomeir and Abou-Donia 1985) or hexanediol Fig. 4 The proportional reduction of VOC with artifcial saliva. (Taher et al. 2009) with appropriate reaction conditions. According to distribute the proportion (Table 1), 16 chemicals were multiplied by corresponding proportion, and results are shown as mean SD (n 3). *Means that the diference values between the Efect of IgY on oral health indexes ± = treatment and control groups are more signifcant compare with Te gingival of rats were red and swollen after 8 chal- another chemicals. The p values are, in order, 0.026, 0.017, 0.023, 0.031, 0.027, 0.035 lenges with P. gingivalis, and the subgingival plaque and bad breath were obvious in most rats. Te scores for GI, PI, and HI decreased signifcantly after treatment with (Table 2, Fig. 6). Te relative contents of hexanal, dimeth- IgY for 4 wks (Fig. 7). For PI, the three treatment groups’ ylfuranone, acetic acid, hexanoic acid, ammonia, and scores were signifcantly lower than the control. For GI, 2-(vinyloxy) propane were signifcantly decreased (Fig. 6) the high-dose group’s scores were lower than the other suggesting that the odor compounds in the oral cavity are treatment groups, and were higher than the positive con- mainly composed of aldehydes, ketone, carboxylic acid, trol group. Tus, the scores of the high-dose group and nitrogen compounds, and alkanes. Te treatment with positive control group were signifcantly decreased. For IgY can efectively inhibit the relative contents of these HI, the high-dose and mid-dose groups’ scores were sig- metabolites produced by P. gingivalis. nifcantly lower than control, while the low-dose group’s Microbial degradation is the main cause of oral malo- scores were higher than the positive control. dor (Bollen and Beikler 2012). Combining the results Te PI and GI were used to diagnose periodontal of the Halimeter and GCMS assays in vitro and in vivo, disease and monitor traditional parameters (Boler- azska et al. 2016). Te HI is an organoleptic parameter. Qiao et al. AMB Expr (2018) 8:176 Page 7 of 12

Table 2 Chemical composition of VOC produced by P. gingivalis in the oral cavity of the rats detected using GCMS No. Retention time CAS no Name Structure Classifcation Proportion of odor (%)a

1 1.3 7664-41-7 Ammonia NH3 Nitrogen compounds (C) 12 2 1.8 926-65-8 2-(Vinyloxy)propane Alkanes (E) 12 O 3 2.5 64-19-7 Acetic acid O Carboxylic acids (A4-1) 12

OH 4 4.3 100-50-5 Cyclohex-3-ene-1-carbaldehyde Aldehydes (A2-1) 1 O

5 5.3 71-41-0 Pentanol HO Alcohols (A1-1) 5

6 6.4 66-25-1 Hexanal O Aldehydes (A2-2) 12

7 8.1 35298-48-7 Dimethylfuranone O Ketones (A3-1) 12

O

8 9-10 111-27-3 n-Hexanol HO Alcohols (A1-2 6 9 14.3 3391-86-4 1-Octen-3-ol OH Alcohols (A1-3) 1

10 14.6 110-93-0 Methyl isohexenyl ketone O Ketones (A3-2) 1

11 15.2 124-13-0 Octyl aldehyde O Aldehydes (A2-3) 1

12 15.5 142-62-1 Hexanoic acid O Carboxylic acids (A4-2) 12

OH 13 16.4 104-76-7 Isooctyl alcohol Alcohols (A1-4) 6 OH 14 17.3 695-06-7 gamma-hexalactone Esters (A5-1) 1 O O

15 17.4 2548-87-0 trans-2-Octen-1-al O Aldehydes (A2-4) 1

16 19.3 30434-65-2 3,4,4-Trimethylcyclopent-2-en-1-one Ketones (A3-3) 1 O

17 23.1 112-31-2 Decyl aldehyde O Aldehydes (A2-5) 1

18 25.4 927-49-1 6-Undecanone O Ketones (A3-4) 1

19 26 540-07-8 Amyl caproate O Esters (A5-2) 1

O

20 28 6064-27-3 Dodecan-6-one O Ketones (A3-5) 1 a According to the contribution to the VOC each chemical’s proportion represents its relative area under the peak without further calibration

Te perception of odor is dependent of the olfactory 4 weeks, these indicators showed statistically signifcant response, the threshold concentration, the strength of the diferences compared with controls, similar to previous odor and the volatility of the molecules. When organo- research (Bostanci et al. 2013). leptic scoring is done, a well-trained clinician determines if the odor samples smells bad or not, giving a score to Efect of IgY on levels of IL‑6 and TNF‑α in serum the intensity (Hu et al. 2007). Tese oral health indica- Abnormally high levels of infammatory factors were tors in rats refected the success of P. gingivalis infec- seen in the control (Fig. 8). After treatment with IgY for tion (Pancer et al. 2016). After treatment with IgY for 4 weeks, the levels of IL-6 and TNF-α in the low-dose Qiao et al. AMB Expr (2018) 8:176 Page 8 of 12

Fig. 5 The reduction of VOC in the oral cavity of the rats. In the oral cavity of the rats, 20 chemicals were detected (Table 2) using GCMS. Negative numbers represent an increase. The contents of VOC reduction are shown as mean with SD error bars (n 3). *Means that the contents of the = control group continued to increase, while the contents of the treatment group decreased signifcantly after administration. The p values are, in order, 0.033, 0.028, 0.043, 0.026, 0.037 compared the control with the treatment groups

Discussion In the present study, IgY showed signifcant inhibitory efects on the bacterial growth of P. gingivalis (Fig. 1) and the production of bacterial metabolites under in vitro (Figs. 2, 3 and 4) and in vivo (Figs. 5 and 6) conditions, as well as moderate inhibitory efects on the development of periodontal infammation and halitosis. IgY both reduced the growth speed of P. gingivalis and decreased the con- centrations of their metabolites to a greater degree than non-specifc IgY, whereas the clinical symptoms of peri- odontal infammation and halitosis were also suppressed (Fig. 7). Tese results suggest that IgY has multiple sup- Fig. 6 The reduction of VOC with a proportion in the oral cavity pressive efects on periodontal infammation and halito- of the rats. Negative numbers represent an increase. According to sis induced by P. gingivalis. distribute the proportion (Table 2), 16 chemicals were multiplied by corresponding proportion, and results are shown as mean SD Te chronic periodontal infammation appears to ± (n 3). *Means that the diference values between the treatment and increase the patients’ risk for cardiovascular diseases = control groups are more signifcant compare with another chemicals. (Genco and Van Dyke 2010), diabetes (Simpson et al. The p values are, in order, 0.036, 0.022, 0.042, 0.035, 0.023, 0.018 2015), cancer (Babic et al. 2015), chronic respiratory dis- eases (Cardoso et al. 2018), adverse pregnancy outcomes (Parihar et al. 2015), and possibly rheumatoid arthri- tis (Kharlamova et al. 2016). Indeed, conventional peri- odontal treatment is often not enough by itself to treat group decreased signifcantly (p < 0.05). At the high- destructive infammation. Developing innovative adjunc- dose, the level of IL-6 was signifcantly (p < 0.01) lower tive therapeutic strategies in chronic periodontitis is as was the level of TNF-α (p < 0.05) compared to con- becoming an urgent need. Some projects have been suc- trol. IL-6 and TNF-α in rat serum promote the pro- cessfully tested to inhibit periodontitis in animal models duction of infammatory factors (Pan et al. 2017). including counteracting immune evasion (McIntosh and Tese markers can be studied both in vitro and in vivo Hajishengallis 2012) or anti- therapy (Cui et al. to learn more about the impact of the treatment. Te 2017). Another approach to treating periodontitis is to experimental rats’ serum stimulated a pro-infamma- the use of agents that promote the resolution of infam- tory response induced by infection of P. gingivalis, while mation by major periodontal . the treatment with IgY decreased the levels of IL-6 and Some experiments revealed specifc IgY can promote TNF-α and shifted the immune response towards an the resolution of infammation in the pathogens infection anti-infammatory response (Bollen and Beikler 2012). Qiao et al. AMB Expr (2018) 8:176 Page 9 of 12

heightened local host response, reimbursing the compro- mised mucosal and provides CF patients with immediate resistance to infection that reduces early colo- nization with P. aeruginosa and hamper the destructive progression into chronic infection (Tomsen et al. 2016). Specifc anti-Staphylococcus aureus IgY between clini- cal and experimental mastitis caused by S. aureus can improve the milk quality and decrease bacterial counts in milk, moreover, the cure rates of mastitis by S. aureus mastitis were higher than that by penicillin (Zhen et al. 2009). Fig. 7 Efect of IgY on rat gingivitis and bad breath induced by P. IgY was a potential alternative for therapy of the gingivalis. The scores for oral health index are shown as mean SD pathogen infection disease, which can stop the major ± (n 3). *Means diferent at p < 0.05. For PI, p 0.027, 0.025 compared periodontal pathogen from breaking gingival tissue and = = the control with the high-dose and low-dose, respectively. For GI, promote the resolution of infammation. In the in vitro p 0.011, 0.032 compared the control with the high-dose and = study, IgY obtained from hens immunized with Solo- low-dose, respectively. For HI, p 0.029, 0.036 compared the control = with the high-dose and low-dose, respectively bacterium moorei inhibited the growth of S. moorei and bioflm formation, meanwhile, in the in vivo study, it had ability to decrease the level of bacteria in the oral cav- ity of mice (Li et al. 2012). Te IgY against P. gingivalis 122k-hemagglutinin A, which was one of the virulence factors of P. gingivalis could be useful in assessing the treatment of periodontal diseases induced by P. gingivalis (Tagawa et al. 2004). Anti-130k haemagglutinin IgY sig- nifcantly inhibited P. gingivalis-associated haemaggluti- nating activity (Hamajima et al. 2007). P. gingivalis were able to evade immune surveillance and avoid clearance, which facilitates local invasions of periodontal tissues; while the host also generated immune responses to the bacterial challenge, which contributed to a bactericidal efect but may also lead to periodontal destruction (Ke et al. 2016), which may develop into periodontitis and halitosis. Treatments of periodontitis and halitosis were Fig. 8 The levels of IL-6 and TNF-α in serum rats. The levels of IL-6 based on mechanical debridement of infectious sites and TNF-α was measured using an ELISA Kit. Data are shown as and pharmacological antibacterial therapy (Huang et al. mean SD (n 3). *Means diferent at p < 0.05, **Means diferent ± = 2013). Te treatments were costly and time consum- at p < 0.01. For IL-6, p 0.007, 0.038 compared the control with the = ing for the patient. Within the limitations of the present high-dose and low-dose, respectively. For TNF-α, p 0.032, 0.045 = compared the control with the high-dose and low-dose, respectively study, in the treatment of oral diseases infected by these pathogens specifc IgY against oral pathogen was shown to be an efective immunotherapeutic substance that P. gingivalis is rigidifed by combining with IgY. diseased. Specifc anti-Vibrio anguillarum IgY adminis- In most cases, halitosis comes from the mouth itself. tration in fsh tissues could reduce bacterial number and About 700 species of microbes collectively populate all infammatory cytokine expression, and enhanced the human mouths. Some studies had shown that the peri- phagocytic activity of against V. anguil- odontal microfora could be involved in the formation of larum in vitro (Li et al. 2014). In the present study, two halitosis, as they had the necessary enzymes and would infammatory cytokine expressions also were inhibited have access to sulfur-containing peptides and amino in the serum of rats by IgY against P. gingivalis (Fig. 8). acids in vivo (Loesche and Kazor 2002). We also per- IgY against Escherichia coli inhibited the growth of the formed metabolomics evaluations to elucidate the molec- pathogen in a dose-dependent manner and enhanced the ular basis of the efects of IgY against P. gingivalis. In the phagocytosis of E. coli (Zhen et al. 2008). In the cystic presence of IgY, a large number of metabolites showed fbrosis (CF) setting, entering Pseudomonas aeruginosa changed concentrations, especially those in the VSC and may be rapidly killed due to IgY against P. aeruginosa VOC biodegradation, which were signifcantly decreased Qiao et al. AMB Expr (2018) 8:176 Page 10 of 12

in a dose-dependent manner. VSC are produced by P. Abbreviations IgY: egg yolk immunoglobulin; VOC: volatile organic compounds; VSC: volatile gingivalis from sulphur amino acids (Bollen and Beikler sulfur compounds; GCMS: gas chromatography mass spectrometry; PI: plaque 2012) in the artifcial saliva. Some researchers have found index; GI: gingival index; HI: halitosis index; IL-6: interleukin-6; TNF-α: tumor that intraoral bacteria metabolise desquamated epithe- necrosis factor α; CF: cystic fbrosis. lial cells and blood cells, leading to the production of Authors’ contributions VSC from cysteine and methionine and thus increasing WQ: Data collection and statistical analysis; FW: Study design, data collection, the VSC values parallel to an increase in the scores for statistical analysis, manuscript preparation and literature search; XX: Literature search and funds collection; SW and JMR: Literature search and funds col- PI and GI (Migliario and Rimondini 2011). Previous work lection; BB and MM: Study design, literature search and funds collection. All showed that tongue cleaning could be used to reduce authors read and approved the fnal manuscript. bacteria as a treatment for halitosis in adults (Outhouse Author details et al. 2006). Essentially any oral site in which microbial 1 College of Food Science and Technology, Shanghai Ocean University, No. 999 accumulation and putrefaction could occur may be an Hucheng Ring Road, Pudong New Area, Shanghai 201306, China. 2 Shanghai 3 origin for oral malodor (Krespi et al. 2006). Te meth- University of Medicine & Health Sciences, Shanghai 201318, China. Shanghai MAXAM Company Limited, Shanghai 200333, China. 4 Co‑Innovation Center ods to reduce halitosis included mechanical and chemo- of Jiangsu Marine Bio‑industry Technology, Huaihai Institute of Technology, therapeutical. Mechanical reduction of malodor and of Lianyungang 222005, China. 5 Department of Food Science, Cornell University, 6 the intraoral bacterial count may be achieved by disrupt- Ithaca, NY 14853‑7201, USA. Laboratory of Quality and Safety Risk Assess- ment for Aquatic Products on Storage and Preservation (Shanghai), Ministry ing the tongue and tooth surface bioflm, thus decreas- of Agriculture, Shanghai 201306, China. ing the production of VSC and other VOC (Aung et al. 2015). Another method, reduction of oral malodor may Acknowledgements The authors would like to thank Shanghai Maxam Ltd. for their material sup- be aided by the use of active antimicrobial compounds port for the project. which decrease the bacterial load and thus decrease the VSC and VOC production (Bollen and Beikler 2012). Competing interests The authors declare that they have no competing interests. However, the reduction was nonspecifc, in other words, all bacterial types were reduced. From an immunological Availability of data and materials point of view, IgY produced by whole- Not applicable. cell lysate had been reported to specifcity prevent H. Consent for publication pylori infection (Horie et al. 2004). It was demonstrated Not applicable. that IgY was highly specifc and had a signifcant efec- Ethics approval and consent to participate tiveness against pathogen. Te advantages of IgY in the All applicable international, national, and/or institutional guidelines for the application included non-invasive during production, care and use of animals were followed. inexpensive, convenience of animal handling and cost Funding efective regarding the high IgY concentration within the Not applicable. egg yolk (Muller et al. 2015). Although the animals and their surroundings are Publisher’s Note specifc pathogen free, and the models of periodontal Springer Nature remains neutral with regard to jurisdictional claims in pub- diseases are typical in this study. However, behavioral dif- lished maps and institutional afliations. ference (e.g., exercise, diet, cleanliness) between individu- Received: 12 July 2018 Accepted: 23 October 2018 als and variability in host responses to bacterial infection may still lead to increased standard deviation of results. Because of drinking and eating after administration, the duration of action of IgY and changes in the oral environ- ments such as pH and salivation may also result in dif- References ferences in the efcacy of IgY. Nonetheless, we always Aung EE, Ueno M, Zaitsu T, Furukawa S, Kawaguchi Y (2015) Efectiveness of three oral hygiene regimens on oral malodor reduction: a randomized strictly control these infuences during the experimental clinical trial. Trials 16:31. https​://doi.org/10.1186/s1306​3-015-0549-9 operation, and try to be uniformity. 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Acta Medica (Hradec Kralove) 59:3–9. https​:// groups showed signifcant anti-infammatory efects sug- doi.org/10.14712​/18059​694.2016.47 gesting that IgY may be an efective treatment for the Bollen CM, Beikler T (2012) Halitosis: the multidisciplinary approach. Int J Oral Sci 4:55–63. https​://doi.org/10.1038/ijos.2012.39 prevention of periodontal infammation and halitosis. Qiao et al. AMB Expr (2018) 8:176 Page 11 of 12

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